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Applied Microbiology and Biotechnology (v.50, #4)
Dialysis cultures by R. Pörtner; H. Märkl (pp. 403-414).
Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a “nutrient-split” feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective.
Microorganism inactivation using high-pressure generation in sealed vessels under sub-zero temperature by K. Hayakawa; Y. Ueno; S. Kawamura; T. Kato; R. Hayashi (pp. 415-418).
In order to test the possibility of utilizing high pressure in bioscience and biotechnology, a simple method for high-pressure generation and its use for microbial inactivation have been studied. When a pressure vessel was filled with water, sealed tightly and cooled to sub-zero temperatures, high pressure was generated in the vessel. The pressure generation was 60 MPa at −5 °C, 103 MPa at −10 °C, and 140 MPa at −15 °C, −20 °C, and −22 °C. The high pressure generated inactivated microorganisms effectively: yeasts (Saccharomyces cerevisiae and Zygosaccharomyces rouxii), bacteria (Lactobacillus brevis and Eschericia coli), and fungi (Aspergillus niger and Aspergillus oryzae) were completely inactivated when stored in sealed vessels −20 °C for 24 h. However, Staphylococcus aureus was only partly inactivated under the same conditions. This method opens up a new application of high pressure for storing, transporting, and sterilizing of foods and biological materials.
Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis by D.-K. Oh; S.-Y. Kim (pp. 419-425).
Candida tropicalis, a strain isolated from the sludge of a factory manufacturing xylose, produced a high xylitol concentration of 131 g/l from 150 g/l xylose at 45 h in a flask. Above 150 g/l xylose, however, volumetric xylitol production rates decreased because of a lag period in cell growth. In fed-batch culture, the volumetric production rate and xylitol yield from xylose varied substantially with the controlled xylose concentration and were maximum at a controlled xylose concentration of 60 g/l. To increase the xylitol yield from xylose, feeding experiments using different ratios of xylose and glucose were carried out in a fermentor. The maximum xylitol yield from 300 g/l xylose was 91% at a glucose/xylose feeding ratio of 15%, while the maximum volumetric production rate of xylitol was 3.98 g l−1 h−1 at a glucose/xylose feeding ratio of 20%. Xylitol production was found to decrease markedly as its concentration rose above 250 g/l. In order to accumulate xylitol to 250 g/l, 270 g/l xylose was added in total, at a glucose/xylose feeding ratio of 15%. Under these conditions, a final xylitol production of 251 g/l, which corresponded to a yield of 93%, was obtained from 270 g/l xylose in 55 h.
Solvent production by Clostridium beijerinckii NRRL B592 growing on different potato media by D. Nimcevic; M. Schuster; J. R. Gapes (pp. 426-428).
Very good solvent formation rates were observed when Clostridium beijerinckii NRRL B592 was cultivated on different whole potato media. The increase in whole potato concentration contributed to the increased final solvent concentrations, while the addition of yeast extract or mineral salts gave negative effects. To obtain good solvent productivities and high final solvent concentrations during batch fermentation, no enzymatic hydrolysis of the potato starch was necessary, indicating high activity of the clostridial amylases produced by the strain applied.
Improving nisin production by increasing nisin immunity/resistance genes in the producer organism Lactococcus lactis by W. S. Kim; R. J. Hall; N. W. Dunn (pp. 429-433).
The effect on nisin production of increasing nisin immunity/resistance genes in Lactococcus lactis subsp. lactis MG1363 was investigated. The 60-kb nisin immunity/resistance plasmid pND300, which was isolated from a non-nisin-producing strain, encodes five genes involved in nisin immunity/resistance, which are very similar to those of the immunity/resistance system encoded by the nisin-production transposon. The introduction of pND300 into MG1363(TnNip) resulted in the construct being able to produce significantly more nisin than the parent MG1363(TnNip). The introduction of pND314, which contains the nisin immunity/resistance genes subcloned into pSA3, into MG1363(TnNip) allowed the strain to grow more rapidly than the parent MG1363(TnNip) with a concomitant increase in the rate of nisin production. This work illustrates that introduction of pND300 and a derivative containing the nisin immunity/resistance system of pND300 into MG1363 (TnNip) can result in significant alterations to the kinetics of nisin production. These observations indicate approaches that may be used successfully to improve the economics of nisin production.
Improved ethanol production by glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae by H. Valadi; C. Larsson; L. Gustafsson (pp. 434-439).
The anaerobic performance of gpd1Δ and gpd2Δ mutants of Saccharomyces cerevisiae was characterized and compared to that of a wild-type strain under well-controlled conditions by using a high-performance bioreactor. There was a 40% reduction in glycerol level in the gpd2Δ mutant compared to the wild-type. Also the gpd1Δ mutant showed a slight decrease in glycerol formation but to a much lesser degree. As a consequence, ethanol formation in the gpd2Δ mutant was elevated by 13%. In terms of growth, the gpd1Δ mutant and the wild-type were indistinguishable. The gpd2Δ mutant, on the other hand, displayed an extended lag phase as well as a reduced growth rate under the exponential phase. Even though glycerol-3-phosphate dehydrogenase 2 (GPD2) is the important enzyme under anaerobic conditions it can, at least in part, be substituted by GPD1. This was indicated by the higher expression level of GPD1 in the gpd2Δ mutant compared to the wild type. These results also show that the cells are able to cope and maintain redox balance under anaerobic conditions even if glycerol formation is substantially reduced, as observed in the gpd2Δ mutant. One obvious way of solving the redox problem would be to make a biomass containing less protein, since most of the excess NADH originates from amino acid biosynthesis. However, the gpd2Δ mutant did not show any decrease in the protein content of the biomass.
Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes by T. Potrawfke; T.-H. Löhnert; K. N. Timmis; R.-M. Wittich (pp. 440-446).
The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction.
Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli by P. J. Punt; I. A. van Gemeren; J. Drint-Kuijvenhoven; J. G. M. Hessing; G. M. van Muijlwijk-Harteveld; A. Beijersbergen; C. T. Verrips; C. A. M. J. J. van den Hondel (pp. 447-454).
The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase (glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed fusion protein were detected in the total protein extracts of these strains.
A Pseudomonas aeruginosa biosensor responds to exposure to ultraviolet radiation by M. O. Elasri; R. V. Miller (pp. 455-458).
We fused the Pseudomonas aeruginosa recA promoter to a promoterless Vibrio fisherilux operon. This recA–lux fusion (pMOE15) was introduced into wild-type P. aeruginosa strain FRD1 and recA expression was monitored by measuring 490-nm light production. The RM4440 strain responded to increasing doses of ultraviolet radiation by an increase in its bioluminescence. RM4440 has the potential to be useful as a biosensor for the presence of DNA-damaging agents in the environment.
Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encoding genes by T. Minetoki; C. Kumagai; K. Gomi; K. Kitamoto; K. Takahashi (pp. 459-467).
The role of the conserved sequence region III in the promoter regions of the amylase-encoding genes amyB, glaA and agdA of Aspergillus oryzae was examined. Introduction of multiple copies of the region III fragment into the agdA promoter resulted in a significant increase in promoter activity at the transcriptional level. This result suggests that the fragment comprising region III consists of one or more cis-acting sequence(s). Moreover, expression of the agdA gene under the control of the improved agdA promoter resulted in efficient overproduction of α -glucosidase, even in the presence of glucose. Thus, overexpression of genes controlled by the improved promoter incorporating region III is possible. Interestingly, expression of the amyB and glaA genes in the transformant was strongly repressed. This result suggests that the trans-acting regulatory protein(s) that interact with region III are common to these amylase genes and that the titration of regulatory protein(s) reduced the expression of the amyB and glaA genes.
Cloning, nucleotide sequencing, and expression of the 3-methylaspartate ammonia-lyase gene from Citrobacter amalonaticus strain YG-1002 by Y. Kato; Y. Asano (pp. 468-474).
The gene coding for 3-methylaspartate ammonia-lyase (3-methylaspartase, MAL, EC 4.3.1.2) from Citrobacter amalonaticus strain YG-1002 (TPU 6323) was cloned onto plasmid pBluescript II KS(+), and the nucleotide sequence of the 1239-bp open reading frame (ORF), consisting of 413 codons, was identified as the mal gene coding for MAL. The predicted polypeptide has 62.5% identity with MAL from the obligate anaerobe, Clostridiumtetanomorphum NCIMB 11547. ORF1, which showed 58.6% and 58.8% identities with subunit E of the glutamate mutases of C. tetanomorphum and Clostridiumcochlearium respectively, was found in the upstream region of the mal gene. An expression plasmid pMALCA3 (5.4 kb), in which the mal gene was expressed under control of the lac promoter on the vector, was constructed. With feeding of 1 mM isopropyl β-d-thiogalactopyranoside, the amount of the enzyme in a cell-free extract of the transformant, E. coli JM109/pMALCA3, was elevated to 51 800 units/l culture, which is about 50-fold that of C. amalonaticus strain YG-1002. It was calculated that the enzyme comprised over 40% of the total extractable cellular proteins. The enzyme produced by the E. coli transformant was purified in a crystalline form and shown to be identical to that of the wild-type strain with respect to specific activity, molecular mass, subunit structure, enzymological properties, and N-terminal amino acid sequences.
Response of fluoranthene-degrading bacteria to surfactants by P. A. Willumsen; U. Karlson; P. H. Pritchard (pp. 475-483).
A prerequisite for surfactant-enhanced biodegradation is that the microorganisms survive, take up substrate and degrade it in the presence of the surfactant. Two Mycobacterium and two Sphingomonas strains, degrading fluoranthene, were investigated for their sensitivity towards non-ionic chemical surfactants. The effect of Triton X-100 and Tween 80 above their critical micelle concentration on mineralization of [14C]-glucose and [14C]-fluoranthene was measured in shaker cultures. Tween 80 had no toxic effect on any of the tested strains. The surfactant inhibited fluoranthene mineralization by the hydrophobic Mycobacterium spp. slightly, but more than doubled that by the two less hydrophobic Sphingomonas strains. Triton X-100 inhibited fluoranthene mineralization by all strains, yet this was more pronounced for the Sphingomonas spp. Both surfactants caused cell wall permeabilization, as shown by transient colouring of surfactant-containing media. Inhibition of glucose mineralization, indicating non-specific toxic effects of Triton X-100, was observed only for the Sphingomonas strains and the toxicity was caused by micelle-to-cell interactions. These strains, however, appeared to recover from initial Triton X-100 toxicity within 50–500 h of exposure. The ratio of surfactant concentration to initial cell density was found to determine critically the bacterial response to surfactants. For both Sphingomonas and Mycobacterium strains, this work indicates that fluoranthene solubilized in surfactant micelles is only partially available for mineralization by the bacteria tested. However, our results suggest that optimal conditions for polycyclic aromatic hydrocarbon mineralization can be developed by selection of the proper surfactant, bacterial strains, cell density and incubation conditions.
Methanogenic and perchloroethylene-dechlorinating activity of anaerobic granular sludge by C. Kennes; M. C. Veiga; L. Bhatnagar (pp. 484-488).
The biodegradation and toxicity of tetrachloroethylene (C2Cl4) and trichloroethylene (C2HCl3) were studied with different anaerobic enrichment cultures using the following electron donors: acetate, propionate, butyrate, methanol, formate and hydrogen. All of them sustained dechlorination except propionate, for which C2Cl4 biodegradation rates were not significant. The best results were obtained with butyrate. Hydrogen appeared to be a relevant electron donor for dechlorination with the present cultures. In the presence of specific inhibitors such as bromoethanesulphonate or molybdate, a slight inhibition of dechlorination was observed. According to dechlorination kinetics, Monod-type behaviour was observed up to 120 μM C2Cl4 or 200 μM C2HCl3 with K s values around 7 μM for both compounds. Dechlorination was partially inhibited at higher concentrations. In contrast, methanogens, or at least methane production, were more sensitive to the presence of chlorinated ethylenes and inhibition of methanogenesis was observed to different extents over all the C2Cl4/C2HCl3 concentration range tested, even at the lowest concentrations.
Repeated application of carvone-induced bacteria to enhance biodegradation of polychlorinated biphenyls in soil by E. S. Gilbert; D. E. Crowley (pp. 489-494).
Carvone, the principal component of spearmint oil, induces biodegradation of polychlorinated biphenyls (PCB) by Arthrobacter sp. strain B1B. This study investigated the effectiveness of the repeated application of carvone-induced bacteria for bioremediation of Aroclor-1242-contaminated soil. Control treatments compared a single inoculation of carvone-induced cells, repeated applications of noninduced cells, and repeated applications of cell-free carvone/fructose medium. The results showed that repeated application of carvone-induced bacteria was the most effective treatment for mineralizing PCB, resulting in 27 ± 6% degradation of Aroclor 1242 after 9 weeks; whereas a single application of cells resulted in no significant degradation. Addition of cell-free, carvone/fructose medium resulted in 10% degradation of PCB, which suggests that this treatment stimulated biodegradation of PCB by the indigenous microflora. The di- and trichlorobiphenyls were the most readily degraded congeners. More highly chlorinated congeners, which had been previously shown to be degraded in liquid culture, were not substantially degraded in soil, indicating that low bioavailability may have limited their degradation. With the development of new technology, which permits automated in situ fermentation and delivery of degrader microorganisms, the repeated application of carvone-induced bacteria may facilitate bioremediation of PCB-contaminated soils.
Effect of ammonia on the anaerobic degradation of protein by a mesophilic and thermophilic biowaste population by C. Gallert; S. Bauer; J. Winter (pp. 495-501).
The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated. For peptone concentrations from 5 g l−1 to 20 g l−1 the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g. 552 mg l−1 day−1 compared to 320 mg l−1 day−1 at 10 g l−1 peptone. The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH3/g peptone in the mesophilic cultures. If 0.5–6.5 g l−1 ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed. The thermophilic biowaste cultures were most active if around 1 g ammonia l−1 was present. Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane. Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a K i (50%) for NH3 of 92, 95 and 88 mg l−1 at 37 °C and 251, 274 and 297 mg l−1 at 55 °C respectively. This indicated that the thermophilic flora tolerated significantly more NH3 than the mesophilic flora. In the mesophilic reactor effluent 4.6 × 108 peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 × 107 cfu/ml was observed.
Electrochemical prevention of marine biofouling on a novel titanium-nitride-coated plate formed by radio-frequency arc spraying by T. Nakayama; H. Wake; K. Ozawa; N. Nakamura; T. Matsunaga (pp. 502-508).
We have developed a new method for forming titanium-nitride(TiN)-coated plates using radio-frequency arc spraying (RFAS). A TiN coating formed by RFAS has been used for electrochemical prevention of marine biofouling. X-ray diffraction and X-ray photoelectron spectroscopy indicate that a TiN composite film containing Ti was formed on a polyethylene terephthalate plate surface when Ti was sprayed by RFAS under atmospheric pressure. A cyclic voltammogram (scan rate 20 mV/s) of the TiN formed by RFAS revealed no oxidative and reductive peak currents in the range −0.6 V to 1.2 V against a saturated silver/silver chloride (Ag/AgCl) electrode. When a potential of 1.0 V against Ag/AgCl was applied to the electrode in seawater, no dissolved Ti was detected. Changes in pH and the chlorine concentration were not observed in this range. In all, only 4.5% of the Vibrio alginolyticus cells attached to the electrode survived when a potential of 0.8 V against Ag/AgCl was applied in seawater for 30 min. In field experiments, attachment of the organisms to the TiN electrode was inhibited by applying an alternating potential of 1.0 V and −0.6 V against Ag/AgCl. The TiN film can be formed by RFAS on large and intricately shaped surfaces, and it is a practical electrode for the electrochemical prevention of fouling of various marine structures.